ABSTRACT The ability to manipulate cellular activities through targeted and precise perturbation promises to dramatically enhance our understanding of biological systems from single molecules to systems-level cell biology. Contrary to the recent explosion of optogenetic modules for electro and chemical signal control, no perturbative tools allowing precise spatiotemporal control of mechanosignaling have been presented so far, despite the importance of mechanosignaling in many developmental, physiological, and pathological processes. The challenge of developing a perturbation toolkit for mechanosignaling stems from the fact that many mechanically-activated processes are localized in space and time and additionally require mechanical loading to become fully activated. To address this, we propose to develop an advanced nanoprobe system with integrated targeting, imaging, and force-generating components. By taking advantage of such multifunctional nanoprobe capabilities, we will systematically investigate the differential effects of biochemical interaction at cell surface, spatial receptor segregation, and mechanical stimulation on regulation of mechanosignaling processes and cellular responses. As initial studies, we propose to investigate interaction and signaling dynamics of neuroligin, integrin, and E-cadherin, key signaling proteins in synaptic function, cell-matrix interactions, and cell-cell junctions, respectively. Ultimately, we aim to provide a platform technology for the systematic investigation of operating principles for a wide range of mechanosensitive proteins, accelerating our understanding of mechanosignaling mechanisms and regulation.